In the intervertebral disc, nucleus pulposus (NP) cells reside in a unique hypoxic niche that imposes metabolic constraints on cells. NP cells exhibit a robust expression of HIF-1? and there is non-canonical control of its turnover and activity by prolyl hydroxylases (PHDs). Based on our recent findings, a major goal of the investigation is to evaluate the mechanisms by which HIF-PHD axis controls NP cell metabolism and if sustained, HIF-1? activity retards age-dependent metabolic and degenerative changes in the disc. In Aim 1 we will test the hypothesis that the HIF-PHD axis is the master regulator of NP cell function in the hypoxic microenvironment through intricate control of their metabolic state. We will determine how HIF-1? controls GLUT-1 and key glycolytic enzyme transcription by ChIP-Seq, mutagenesis and silencing or overexpression approaches. The role of HIF-1 in controlling metabolic flux will be delineated by measuring the fate of [1-2- 13C2]-glucose and [U-13C5]-glutamine in NP cells from young and old rats. We will delete GLUT-1 using NP specific FoxA2-Cre and Shh-CreERT2-Cre mice. Finally, we will use NP cells isolated from human degenerated tissues to determine how disease severity alters the expression of GLUT-1 and HIF-1 dependent metabolic targets. In Aim 2, we will test the hypothesis that pH homeostasis in glycolytic NP cells is regulated by HIF-1?- dependent molecular circuit comprising the lactate transporter, MCT4, its accessory protein basigin and plasma membrane associated carbonic anhydrase (CA) 9 and 12. We have shown that HIF-1-dependent expression of CA9 and 12 play a critical role in cytosolic pH maintenance through HCO3- recycling. We will determine mechanisms by which HIF-1 controls MCT4 and basigin expression. We will delineate the functional role of MCT4 in cytosolic clearance of glycolytic end products, lactate and H+. Using MCT4 knockout mice, we will ascertain if perturbation of pH homeostasis compromises disc health with aging. Finally, using human degenerated tissues we will determine how HIF-1? activity and disease severity alters expression of MCT4, basigin and CA9/12. In Aim 3 we will test the hypothesis that increasing HIF-1? activity rescues NP cells from age-dependent disc degeneration through maintenance of glycolytic metabolism and pH homeostasis. We showed that PHD3 controls HIF-1? activity and lack of PHD3 in vivo promotes NP degeneration. We will conditionally overexpress HIF-1? in the NP of PHD3-/- mice and examine the age dependent changes in disc phenotype. We will study the influence of restored HIF-1? activity on expression of key metabolic and pH homeostatic regulators. Finally, we will determine if HIF-1? overexpression alone slows down the progression of age-dependent disc degeneration. The studies are first-of-a-kind in field of disc research and will provide insights into the unique metabolic control of NP cells by the HIF-PHD circuit. The investigations will generate metabolic biomarkers of healthy and degenerating NP cells as well as novel druggable targets. The outcomes will provide rationale for use of PHD inhibitors (in clinical trials for treating anemia) to control disc disease.